Enzymatic production of D-allulose

ABSTRACT

The current disclosure provides a process for enzymatically converting a saccharide into allulose. The invention also relates to a process for preparing allulose where the process involves converting fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P), catalyzed by allulose 6-phosphate 3-epimerase (A6PE), and converting the A6P to allulose, catalyzed by allulose 6-phosphate phosphatase (A6PP).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 16/468,916, filed on Dec. 14, 2017, which is a 371 application ofInternational Application No. PCT/US/2017/066298, which claims priorityto U.S. Application No. 62/434,033, filed on Dec. 14, 2016, which isincorporated herein by reference.

SEQUENCE LISTING

The Sequence Listing submitted herewith is an ASCII text file(2020-03-13_Sequence_Listing.text, created on Mar. 12, 2020, 16384bytes) via EFS-Web is hereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to preparation of the sugar D-allulose. Morespecifically, the invention relates to methods of preparing D-alluloseby enzymatically converting saccharides (e.g., polysaccharides,oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose)into D-allulose.

BACKGROUND OF THE INVENTION

D-allulose (also known as D-psicose) (allulose hereafter) is alow-calorie, natural sweetener that has 70% the sweetness of sucrose,but only 10% of the calories. It is a naturally occurring monosaccharidehexose that is present in only small amounts in wheat and other plants.Allulose was approved as a food additive by the Food and DrugAdministration (FDA) in 2012, which designated it as generallyrecognized as safe (GRAS). However, due to allulose's high sellingprices, its use as a sweetener has been limited. Allulose boasts amyriad of health benefits: it is low-calorie (10% of sucrose); it has avery low glycemic index of 1; it is fully absorbed in the smallintestine but not metabolized and instead secreted in urine and feces;it helps regulate blood sugar by inhibiting alpha-amylase, sucrase andmaltase; and it has similar functionality in foods and beverages assucrose. As such, allulose clearly has a variety of applications in thefood and beverage industries.

Currently allulose is produced predominantly through the enzymaticisomerization of fructose (WO 2014049373). Overall, the method suffersbecause of higher feedstock cost, the costly separation of allulose fromfructose, and relatively low product yields.

There is a need to develop a cost-effective synthetic pathway forhigh-yield allulose production where at least one step of the processinvolves an energetically favorable chemical reaction. Furthermore,there is a need for production process where the process steps can beconducted in one tank or bioreactor. There is also a need for a processof allulose production that can be conducted at a relatively lowconcentration of phosphate, where phosphate can be recycled, and/or theprocess does not require using adenosine triphosphate (ATP) as a sourceof phosphate. There is also a need for an allulose production pathwaythat does not require the use of the costly nicotinamide adenosinedinucleotide (NAD(H)) coenzyme in any of the reaction steps.

SUMMARY OF THE INVENTION

The inventions described herein relate to processes for preparingallulose. In various aspects, the processes involve converting fructose6-phosphate (F6P) to allulose 6-phosphate (A6P), catalyzed by allulose6-phosphate 3-epimerase (A6PE); and converting the A6P to allulose,catalyzed by allulose 6-phosphate phosphatase (A6PP). The inventionsalso relate to allulose prepared by any of the processes describedherein.

In some aspects of the invention, a process for preparing allulose alsoinvolves the step of converting glucose 6-phosphate (G6P) to the F6P,where the step is catalyzed by phosphoglucoisomerase (PGI). In otheraspects, a process for allulose synthesis also includes the step ofconverting glucose 1-phosphate (G1P) to the G6P, and this conversionstep is catalyzed by phosphoglucomutase (PGM).

In various aspects, a process for preparing allulose can involveconverting a saccharide to the G1P, catalyzed by at least one enzyme;converting G1P to G6P, catalyzed by phosphoglucomutase (PGM); convertingG6P to F6P, catalyzed by phosphoglucoisomerase (PGI); converting F6P toallulose 6-phosphate (A6P), catalyzed by A6PE; and converting the A6Pproduced to allulose, catalyzed by A6PP.

The saccharides used in any of the processes can be selected from thegroup consisting of a starch or its derivative, cellulose or itsderivative, and sucrose. The starch or its derivative can be amylose,amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, orglucose. In some aspects of the invention, a process for preparingallulose involves converting starch to a starch derivative by enzymatichydrolysis or by acid hydrolysis of starch. In other aspects, a starchderivative can be prepared by enzymatic hydrolysis of starch catalyzedby isoamylase, pullulanase, alpha-amylase, or a combination of two ormore of these enzymes. A process for preparing allulose, in certainaspects, can also involve adding 4-glucan transferase (4GT).

In various aspects, a process for preparing allulose can involveconverting fructose to the F6P, catalyzed by at least one enzyme;converting F6P to allulose 6-phosphate (A6P) catalyzed by A6PE; andconverting the A6P produced to allulose, catalyzed by A6PP. In otherembodiments, allulose production process involves converting sucrose tothe fructose, catalyzed by at least one enzyme; converting fructose tothe F6P, catalyzed by at least one enzyme; converting F6P to allulose6-phosphate (A6P) catalyzed by A6PE; and converting the A6P produced toallulose, catalyzed by A6PP.

In other aspects of the invention, G6P to be used in a process forpreparing allulose can be generated by converting glucose to the G6P,catalyzed by at least one enzyme. Glucose can in turn be produced byconverting sucrose to glucose, catalyzed by at least one enzyme.

In other aspects of the invention, the steps of a process for preparingallulose are conducted ATP-free, NAD(H)-free, at a phosphateconcentration from about 0 mM to about 150 mM, the phosphate isrecycled, and/or at least one step of the process involves anenergetically favorable chemical reaction.

BRIEF DESCRIPTION OF THE FIGURES

These drawings illustrate certain aspects of some of the embodiments ofthe invention, and should not be used to limit or define the invention.

FIG. 1 is a schematic diagram illustrating an enzymatic pathwayconverting fructose 6-phosphate to allulose 6-phosphate and then toallulose.

FIG. 2 is a schematic diagram illustrating an enzymatic pathwayconverting starch or its derived products to allulose. The followingabbreviations are used: αGP, alpha-glucan phosphorylase or starchphosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; IA,isoamylase; PA, pullulanase; MP, maltose phosphorylase; PPGK,polyphosphate glucokinase.

FIG. 3 shows an enzymatic pathway converting cellulose or its derivedproducts to allulose. CDP, cellodextrin phosphorylase; CBP, cellobiosephosphorylase; PPGK, polyphosphate glucokinase; PGM, phosphoglucomutase;PGI, phosphoglucoisomerase.

FIG. 4 is a schematic diagram illustrating an enzymatic pathwayconverting fructose to allulose. PPFK, polyphosphate fructokinase.

FIG. 5 is a schematic diagram illustrating an enzymatic pathwayconverting glucose to allulose. PPGK, polyphosphate glucokinase; PGI,phosphoglucoisomerase.

FIG. 6 shows an enzymatic pathway converting sucrose or its derivedproducts to allulose. SP, sucrose phosphorylase; PPFK, polyphosphatefructokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase.

FIG. 7 shows the Reaction Gibbs Energy between intermediates based onformation Gibbs energy for the conversion of glucose 1-phosphate toallulose.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides enzymatic pathways, or processes, forsynthesizing allulose with a high product yield, while greatlydecreasing the product separation costs and allulose production costs.

The invention relates to a process for preparing allulose where theprocess involves converting fructose 6-phosphate (F6P) to allulose6-phosphate (A6P) catalyzed by an epimerase and converting the A6Pproduced to allulose catalyzed by a phosphatase (e.g., allulose6-phosphate phosphatase, A6PP). This process is generally shown inFIG. 1. In certain embodiments, the epimerase that catalyzes theconversion of F6P to A6P is allulose 6-phosphate 3-epimerase (A6PE).

Epimerases that convert F6P to A6P may be used in a process of theinvention. Epimerases are also capable of converting A6P to F6P. In someaspects of the invention, epimerases suitable for use in the processesto convert F6P to A6P comprise an amino acid sequence that has a degreeof identity to the amino acid sequence of SEQ ID NOs.: 3 or 6, of atleast 45%, more preferably at least 50%, more preferably at least 55%,more preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, at least 91%, at least 92%, at least 93%, or at least 94%,most preferably at least 95%, and even most preferably at least 96, 97,98, 99 or 100%. The suitable epimerases are encoded by a polynucleotidecomprising a nucleotide sequence that has a degree of identity to thenucleotide sequence of SEQ ID NOS.: 1, 2, 4, and 5; of at least 30%,preferably at least 35%, more preferably at least 40%, more preferablyat least 45%, more preferably at least 50%, more preferably at least55%, more preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96, 97, 98, 99 or 100%.

Examples of A6PEs include, but are not limited to the followingproteins, identified by UNIPROT ID numbers: D9TQJ4, A0A090IXZ8, andP32719. Of these, D9TQJ4 and A0A090IXZ8 are obtained from thermophilicorganisms. P32719 is obtained from a mesophilic organism. P32719 is 53%identical to A0A090IXZ8 and 55% identical to D9TQJ4, and each proteincatalyzes the epimerization of F6P to A6P. Furthermore, A0A090IXZ8 is45% identical to D9TQJ4. Conversely, other epimerase proteins identifiedby UNIPROT ID numbers: A0A101D823, R1AXD6, A0A150LBU8, A0A023CQG9, andH1XWY2, which have a degree of identity to D9TQJ4 of 45% or less do notcatalyze the epimerization of F6P to A6P.

In some aspects of the invention, epimerases suitable for use in theprocesses to convert F6P to A6P utilize a divalent metal cofactor:preferably, but not limited to, cobalt. In further aspects of theinvention the epimerase contains but is not limited to containing an(α/(β)₈-barrel domain for catalysis; additionally but not limited tocontaining a phosphate binding domain including a Ser at the end of the7^(th) β-strand of the barrel, a Ser at the end of the 8^(th) β-strandof the barrel, and a Gly in the active site loop; additionally but notlimited to containing a metal binding domain including a His in the2^(nd) and 3^(rd) β-strands of the barrel; additionally but not limitedto containing an Asp in the 2^(nd) and 7^(th) β-strand of the barrel toact as the acid/base catalyst for 1,1 proton transfer, and additionallybut not limited to containing a His-hydrophobic residue-Asp signature inthe 2^(nd) β-strand of the barrel where the His is utilized in metalbinding and the Asp for acid/base catalysis. These features are known inthe art, and are referenced in, for example, Chan et al., StructuralBasis for Substrate Specificity in Phosphate Binding(beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase fromEscherichia coli K-12. Biochemistry. 2008; 47 (36); 9608-9617.Preferably, an epimerase for use in the processes of the inventioncontains an (α/β)₈-barrel domain for catalysis, a Ser at the end of the7th β-strand of the barrel, a Ser at the end of the 8th β-strand of thebarrel, a Gly in the active site loop, a His in the 2nd and 3rdβ-strands of the barrel, an Asp in the 2nd and 7th β-strand of thebarrel, and a His-hydrophobic residue-Asp signature in the 2nd β-strandof the barrel.

Processes of the invention use phosphatases that convert A6P to allulose(D-allulose). In some aspects of the invention, phosphatases suitablefor the process to convert A6P to allulose comprise an amino acidsequence that has a degree of identity to the amino acid sequence of SEQID NO.: 9, of at least 45%, more preferably at least 50%, morepreferably at least 55%, more preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95%, at least91%, at least 92%, at least 93%, or at least 94%, and even mostpreferably at least 96, 97, 98, 99 or 100%. The suitable epimerases areencoded by a polynucleotide comprising a nucleotide sequence that has adegree of identity to the nucleotide sequence of SEQ ID NOS.: 7 and 8;of at least 30%, preferably at least 35%, more preferably at least 40%,more preferably at least 45%, more preferably at least 50%, morepreferably at least 55%, more preferably at least 60%, more preferablyat least 65%, more preferably at least 70%, more preferably at least75%, more preferably at least 80%, more preferably at least 85%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 96, 97, 98, 99 or 100%.

Examples of A6PPs include, but are not limited to the followingproteins, identified by UNIPROT ID numbers: A3DC21, Q5LGR4, and Q89ZR1.A3DC21 is 46% identical to Q5LGR4 and 45% identical to Q89ZR1, and eachprotein catalyzes the specific dephosphorylation of A6P to allulose.Conversely, other phosphatases from the haloacid dehydrogenase superfamily, proteins identified by UNIPROT ID numbers: H0UQ29, Q67LU4,A0A0K6IPM3, C8WSJ0, A0A151YX61, and others, which are less than 45%identical to A3DC21 do not catalyze the specific dephosphorylation ofA6P to allulose.

Phosphatases to convert A6P to allulose, suitable for use in theprocesses of the invention are specific to allulose 6-phosphate. As usedherein, specific to allulose 6-phosphate refers to having a higherspecific activity on allulose 6-phosphate compared to glucose1-phosphate, glucose 6-phosphate, or fructose 6-phosphate.

Phosphatases to convert A6P to allulose utilize a divalent metalcofactor: preferably magnesium. In further aspects of the invention thephosphatase contains but is not limited to containing a Rossmanoid folddomain for catalysis; additionally but not limited to containing a C1capping domain for substrate specificity; additionally but not limitedto containing a DxD signature in the 1^(st) β-strand of the Rossmanoidfold for coordinating magnesium where the second Asp is a generalacid/base catalyst; additionally but not limited to containing a Thr orSer at the end of the 2^(nd) β-strand of the Rossmanoid fold that helpsstability of reaction intermediates; additionally but not limited tocontaining a Lys at the N-terminus of the a-helix C-terminal to the3^(rd) β-strand of the Rossmanoid fold that helps stability of reactionintermediates; and additionally but not limited to containing a GDxxxDsignature at the end of the 4^(th) β-strand of the Rossmanoid fold forcoordinating magnesium. These features are known in the art and arereferenced in, for example, Burroughs et al., Evolutionary Genomics ofthe HAD Superfamily: Understanding the Structural Adaptations andCatalytic Diversity in a Superfamily of Phosphoesterases and AlliedEnzymes. J. Mol. Biol. 2006; 361; 1003-1034. Preferably, a phosphataseto convert A6P to allulose used in the processes of the inventioncontains a Rossmanoid fold domain for catalysis, a C1 capping domain,DxD signature in the 1st β-strand of the Rossmanoid fold, a Thr or Serat the end of the 2nd β-strand of the Rossmanoid fold, a Lys at theN-terminus of the α-helix C-terminal to the 3rd β-strand of theRossmanoid fold, and a GDxxxD signature at the end of the 4th β-strandof the Rossmanoid fold.

In some embodiments, a process for preparing allulose according to theinvention also includes the step of enzymatically converting glucose6-phosphate (G6P) to the F6P, and this step is catalyzed byphosphoglucoisomerase (PGI). In other embodiments, the process forpreparing allulose additionally includes the step of converting glucose1-phosphate (G1P) to the G6P, where the step is catalyzed byphosphoglucomutase (PGM). In yet further embodiments, alluloseproduction process also includes the step of converting a saccharide tothe G1P that is catalyzed at least one enzyme.

Therefore, a process for preparing allulose according to the inventioncan, for example, include the following steps: (i) converting asaccharide to glucose 1-phosphate (G1P) using one or more enzymes; (ii)converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii)converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9);(iv) converting F6P to A6P via A6PE, and (v) converting A6P to allulosevia A6PP. An example of the process where the saccharide is starch isshown in FIG. 2.

Typically, the ratios of enzyme units used in the disclosed process are1:1:1:1:1 (αGP:PGM:PGI:A6PE:A6PP). To optimize product yields, theseratios can be adjusted in any number of combinations. For example, aratio of 3:1:1:1:1 can be used to maximize the concentration ofphosphorylated intermediates, which will result in increased activity ofthe downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be usedto maintain a robust supply of phosphate for αGP, which will result inmore efficient phosphorolytic cleavage of alpha-1,4-glycosidic bonds. Aratio of enzymes, for example, 3:1:1:1:3 can be used to further increasethe reaction rate. Therefore, the enzyme ratios, including otheroptional enzymes discussed below, can be varied to increase theefficiency of allulose production. For example, a particular enzyme maybe present in an amount about 2×, 3×, 4×, 5×, etc. relative to theamount of other enzymes.

One of the important advantages of the processes is that the processsteps can be conducted in one bioreactor or reaction vessel.Alternatively, the steps can also be conducted in a plurality ofbioreactors, or reaction vessels, that are arranged in series.

Phosphate ions produced by dephosphorylation of A6P can then be recycledin the process step of converting a saccharide to G1P, particularly whenall process steps are conducted in a single bioreactor or reactionvessel. The ability to recycle phosphate in the disclosed processesallows for non-stoichiometric amounts of phosphate to be used, whichkeeps reaction phosphate concentrations low. This affects the overallpathway and the overall rate of the processes, but does not limit theactivity of the individual enzymes and allows for overall efficiency ofthe allulose making processes.

For example, reaction phosphate concentrations can range from about 0 mMto about 300 mM, from about 0 mM to about 150 mM, from about 1 mM toabout 50 mM, preferably from about 5 mM to about 50 mM, or morepreferably from about 10 mM to about 50 mM. For instance, the reactionphosphate concentration can be about 0.1 mM, about 0.5 mM, about 1 mM,about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM,or about 55 mM.

Therefore, low phosphate concertation results in decreased productioncosts due to low total phosphate and thus lowered cost of phosphateremoval. It also prevents inhibition of the A6PP by high concentrationsof free phosphate and decreases the potential for phosphate pollution.

Furthermore, the processes disclosed herein can be conducted withoutadded ATP as a source of phosphate, i.e., ATP-free. The processes canalso be conducted without having to add NAD(H), i.e., NAD(H)-free. Otheradvantages also include the fact that at least one step of the disclosedprocesses for making allulose involves an energetically favorablechemical reaction (FIG. 7).

Examples of the enzymes used to convert a saccharide to G1P includealpha-glucan phosphorylase (αGP, EC 2.4.1.1 which also includesmaltodextrin phosphorylase, starch phosphorylase, glycogenphosphorylase, and other α-1,4 glycosidic bond degradingphosphorylases), maltose phosphorylase (MP, EC 2.4.1.8), cellodextrinphosphorylase (CDP, EC 2.4.1.49), cellobiose phosphorylase (CBP, EC2.4.1.20), cellulose phosphorylase, sucrose phosphorylase (SP, EC2.4.1.7), and a combination thereof. The choice of the enzyme or enzymecombination depends on the saccharide used in the process.

The saccharides used for generating G1P can be polysaccharides,oligosaccharides, and/or disaccharides. For example, the saccharide canbe starch, one or more derivatives of starch, cellulose, one or morederivatives of cellulose, sucrose, one or more derivatives of sucrose,or a combination thereof.

Starch is the most widely used energy storage compound in nature and ismostly stored in plant seeds. Natural starch contains linear amylose andbranched amylopectin. Examples of starch derivatives include amylose,amylopectin, soluble starch, amylodextrin, maltodextrin, maltose,fructose, and glucose. Examples of cellulose derivatives includepretreated biomass, regenerated amorphous cellulose, cellodextrin,cellobiose, fructose, and glucose. Sucrose derivatives include fructoseand glucose.

The derivatives of starch can be prepared by enzymatic hydrolysis ofstarch or by acid hydrolysis of starch. Specifically, the enzymatichydrolysis of starch can be catalyzed or enhanced by isoamylase (IA, EC.3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds; pullulanase (PA, EC.3.2.1.41), which hydrolyzes α-1,6-glucosidic bonds;4-α-glucanotransferase (4GT, EC. 2.4.1.25), which catalyzes thetransglycosylation of short maltooligosaccharides, yielding longermaltooligosaccharides; or alpha-amylase (EC 3.2.1.1), which cleavesα-1,4-glucosidic bonds.

Furthermore, derivatives of cellulose can be prepared by enzymatichydrolysis of cellulose catalyzed by cellulase mixtures, by acids, or bypretreatment of biomass.

In certain embodiments, the enzymes used to convert a saccharide to G1Pcontain αGP. In this step, when the saccharides include starch, the G1Pis generated from starch by αGP; when the saccharides contain solublestarch, amylodextrin, or maltodextrin, the G1P is produced from solublestarch, amylodextrin, or maltodextrin by αGP.

When the saccharides include maltose and the enzymes contain maltosephosphorylase, the G1P is generated from maltose by maltosephosphorylase. If the saccharides include sucrose, and enzymes containsucrose phosphorylase, the G1P is generated from sucrose by sucrosephosphorylase.

In yet another embodiment, when the saccharides include cellobiose, andthe enzymes contain cellobiose phosphorylase, the G1P is generated fromcellobiose by cellobiose phosphorylase.

In an additional embodiment, when the saccharides contain cellodextrinsand the enzymes include cellodextrin phosphorylase, the G1P is generatedfrom cellodextrins by cellodextrin phosphorylase.

In an alternative embodiment of converting a saccharide to G1P, when thesaccharides include cellulose, and enzymes contain cellulosephosphorylase, the G1P is generated from cellulose by cellulosephosphorylase.

According to the invention, allulose can also be produced from fructose.An example of the process is shown in FIG. 4. For example, the processinvolves generating F6P from fructose and polyphosphate catalyzed bypolyphosphate fructokinase (PPFK); converting F6P to A6P catalyzed byA6PE; and converting A6P to allulose catalyzed by A6PP. The fructose canbe produced, for example, by an enzymatic conversion of sucrose.

In other embodiments, allulose can be produced from sucrose. An exampleof such process is shown in FIG. 6. The process provides an in vitrosynthetic pathway that includes the following enzymatic steps:generating G1P from sucrose and free phosphate catalyzed by sucrosephosphorylase (SP); converting G1P to G6P catalyzed by PGM; convertingG6P to F6P catalyzed by PGI; converting F6P to A6P catalyzed by A6PE;and converting A6P to allulose catalyzed by A6PP.

The phosphate ions generated when A6P is converted to allulose can thenbe recycled in the step of converting sucrose to G1P. Additionally, asshown in FIG. 6, PPFK and polyphosphate can be used to increase alluloseyields by producing F6P from fructose generated by the phosphorolyticcleavage of sucrose by SP.

In some embodiments, a process for preparing allulose includes thefollowing steps: generating glucose from polysaccharides andoligosaccharides by enzymatic hydrolysis or acid hydrolysis, convertingglucose to G6P catalyzed by at least one enzyme, generating fructosefrom polysaccharides and oligosaccharides by enzymatic hydrolysis oracid hydrolysis, and converting fructose to G6P catalyzed by at leastone enzyme. Examples of the polysaccharides and oligosaccharides areenumerated above.

In other embodiments, G6P is produced from glucose and sodiumpolyphosphate by polyphosphate glucokinase.

The present disclosure provides processes for converting saccharides,such as polysaccharides and oligosaccharides in starch, cellulose,sucrose and their derived products, to allulose. In certain embodiments,artificial (non-natural) ATP-free enzymatic pathways are provided toconvert starch, cellulose, sucrose, and their derived products toallulose using cell-free enzyme cocktails.

As shown above, several enzymes can be used to hydrolyze starch toincrease the G1P yield. Such enzymes include isoamylase, pullulanase,and alpha-amylase. Corn starch contains many branches that impede αGPaction. Isoamylase can be used to de-branch starch, yielding linearamylodextrin. Isoamylase-pretreated starch can result in a higher F6Pconcentration in the final product. Isoamylase and pullulanase cleavealpha-1,6-glycosidic bonds, which allows for more complete degradationof starch by alpha-glucan phosphorylase. Alpha-amylase cleavesalpha-1,4-glycosidic bonds, therefore alpha-amylase is used to degradestarch into fragments for quicker conversion to allulose.

As shown in FIG. 2, maltose phosphorylase (MP) can be used to increaseallulose yields by phosphorolytically cleaving the degradation productmaltose into G1P and glucose. Alternatively, 4-glucan transferase (4GT)can be used to increase allulose yields by recycling the degradationproducts glucose, maltose, and maltotriose into longermaltooligosaccharides; which can be phosphorolytically cleaved by αGP toyield G1P.

Additionally, cellulose is the most abundant bio resource and is theprimary component of plant cell walls. Non-food lignocellulosic biomasscontains cellulose, hemicellulose, and lignin as well as other minorcomponents. Pure cellulose, including Avicel (microcrystallinecellulose), regenerated amorphous cellulose, bacterial cellulose, filterpaper, and so on, can be prepared via a series of treatments. Thepartially hydrolyzed cellulosic substrates include water-insolublecellodextrins whose degree of polymerization is more than 7,water-soluble cellodextrins with degree of polymerization of 3-6,cellobiose, glucose, and fructose.

In certain embodiments, cellulose and its derived products can beconverted to allulose through a series of steps. An example of suchprocess is a shown in FIG. 3. The process provides an in vitro syntheticpathway that involves the following steps: generating G1P fromcellodextrin and cellobiose and free phosphate catalyzed by cellodextrinphosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively;converting G1P to G6P catalyzed by PGM; converting G6P to F6P catalyzedby PGI; converting F6P to A6P catalyzed by A6PE; and converting A6P toallulose catalyzed by A6PP. In this process, the phosphate ions can berecycled by the step of converting cellodextrin and cellobiose to G1P.

Several enzymes may be used to hydrolyze solid cellulose towater-soluble cellodextrins and cellobiose. Such enzymes includeendoglucanase and cellobiohydrolase, but not including beta-glucosidase(cellobiase).

Prior to cellulose hydrolysis and G1P generation, cellulose and biomasscan be pretreated to increase their reactivity and decrease the degreeof polymerization of cellulose chains. Cellulose and biomasspretreatment methods include dilute acid pretreatment, cellulosesolvent-based lignocellulose fractionation, ammonia fiber expansion,ammonia aqueous soaking, ionic liquid treatment, and partiallyhydrolyzed by using concentrated acids, including hydrochloric acid,sulfuric acid, phosphoric acid and their combinations.

In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK)can be added to the process, thus increasing yields of allulose byphosphorylating the degradation product glucose to G6P, as shown in FIG.3.

In other embodiments, allulose can be generated from glucose. An exampleof such process is shown in FIG. 5. The process involves the steps ofgenerating G6P from glucose and polyphosphate catalyzed by polyphosphateglucokinase (PPGK); converting G6P to F6P catalyzed by PGI; convertingF6P to A6P catalyzed by an enzyme; and converting A6P to allulosecatalyzed by A6PP.

Any suitable biological buffer known in the art can be used in a processof the invention, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma,etc. The reaction buffer for all embodiments can have a pH ranging from5.0-8.0. More preferably, the reaction buffer pH can range from about6.0 to about 7.3. For example, the reaction buffer pH can be 6.0, 6.2,6.4, 6.6, 6.8, 7.0, 7.2, or 7.3.

The reaction buffer can also contain key metal cations. Examples of themetal ions include Mg²⁺, Co²⁺, and Zn²⁺.

The reaction temperature at which the process steps are conducted canrange from 37-85° C. More preferably, the steps can be conducted at atemperature ranging from about 40° C. to about 70° C. The temperaturecan be, for example, about 40° C., about 45° C., about 50° C., about 55°C., or about 60° C. Preferably, the reaction temperature is about 50° C.

The reaction time of the disclosed processes can be adjusted asnecessary, and can range from about 8 hours to about 48 hours. Forexample, the reaction time can be about 16 hours, about 18 hours, about20 hours, about 22 hours, about 24 hours, about 26 hours, about 28hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours,about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46hours, or about 48 hours. More preferably, the reaction time is about 24hours.

The processes according to the invention can achieve high yields due tothe very favorable equilibrium constant for the overall reaction.Theoretically, up to 99% yields can be achieved if the starting materialis completely converted to an intermediate.

Processes of the invention use low-cost starting materials and reduceproduction costs by decreasing costs associated with the feedstock andproduct separation. Starch, cellulose, sucrose and some of theirderivatives are less expensive feedstocks than, for example, fructose.When allulose is produced from fructose, yields are lower than in thepresent invention, and allulose must be separated from fructose viachromatography, which leads to higher production costs.

Also, the step of converting A6P to allulose according to the inventionis an irreversible phosphatase reaction, regardless of the feedstock.Therefore, allulose is produced with a very high yield while effectivelyminimizing the subsequent product separation costs.

In contrast to cell-based manufacturing methods, the invention involvesa cell-free preparation of allulose, has relatively high reaction ratesdue to the elimination of the cell membrane, which often slows down thetransport of substrate/product into and out of the cell. It also has afinal product free of nutrient-rich fermentation media/cellularmetabolites.

EXAMPLES

Materials and Methods

Chemicals

All chemicals, including corn starch, soluble starch, maltodextrins,maltose, glucose, filter paper were reagent grade or higher andpurchased from Sigma-Aldrich (St. Louis, Mo., USA) or Fisher Scientific(Pittsburgh, Pa., USA), unless otherwise noted. Restriction enzymes, T4ligase, and Phusion DNA polymerase were purchased from New EnglandBiolabs (Ipswich, Mass., USA). Oligonucleotides were synthesized eitherby Integrated DNA Technologies (Coralville, Iowa, USA) or Eurofins MWGOperon (Huntsville, Ala., USA). The nucleotide sequence, SEQ ID NO 1,encodes for Thermophilic A6PE from Thermoanaerobacteriumthermosaccharolyticum (UNIPROT ID D9TQJ4). SEQ ID NO 2 is a codonoptimized version of that nucleotide sequence. SEQ ID NO 3 is the aminoacid sequence for the enzyme. The nucleotide sequence SEQ ID NO 4encodes for Thermophilic A6PE from Bacillus thermoamylovorans (UNIPROTID A0A090IXZ8). SEQ ID NO 5 is a codon optimized version of thatnucleotide sequence. SEQ ID NO 6 is the amino acid sequence for theenzyme. The nucleotide sequence SEQ ID NO 7 encodes for ThermophilicA6PP from Clostridium thermocellum (UNIPROT ID A3DC21). SEQ ID NO 8 is acodon optimized version of the nucleotide sequence. SEQ ID NO 9 is theamino acid sequence corresponding to the enzyme. Regenerated amorphouscellulose used in enzyme purification was prepared from Avicel PH105(FMC BioPolymer, Philadelphia, Pa., USA) through its dissolution andregeneration, as described in: Ye et al., Fusion of a family 9cellulose-binding module improves catalytic potential of Clostridiumthermocellum cellodextrin phosphorylase on insoluble cellulosel. Appl.Microbiol. Biotechnol. 2011; 92:551-560. Escherichia coli Sig10(Sigma-Aldrich, St. Louis, Mo., USA) was used as a host cell for DNAmanipulation and E. coli BL21 (DE3) (Sigma-Aldrich, St. Louis, Mo., USA)was used as a host cell for recombinant protein expression. ZYM-5052media including either 100 mg L⁻¹ ampicillin or 50 mg L⁻¹ kanamycin wasused for E. coli cell growth and recombinant protein expression.Cellulase from Trichoderma reesei (Catalog number: C2730) andpullulanase (Catalog number: P1067) were purchased from Sigma-Aldrich(St. Louis, Mo., USA) and produced by Novozymes (Franklinton, N.C.,USA). Maltose phosphorylase (Catalog number: M8284) was purchased fromSigma-Aldrich.

Production and Purification of Recombinant Enzymes

The E. coli BL21 (DE3) strain harboring a protein expression plasmid wasincubated in a 1-L Erlenmeyer flask with 100 mL of ZYM-5052 mediacontaining either 100 mg L⁻¹ ampicillin or 50 mg L⁻¹ kanamycin. Cellswere grown at 30° C. with rotary shaking at 220 rpm for 16-24 hours. Thecells were harvested by centrifugation at 12° C. and washed once witheither 20 mM HEPES (pH 7.5) containing 50 mM NaCl and 5 mM MgCl₂ (heatprecipitation and cellulose-binding module) or 20 mM HEPES (pH 7.5)containing 300 mM NaCl and 5 mM imidazole (Ni purification). The cellpellets were re-suspended in the same buffer and lysed byultra-sonication (Fisher Scientific Sonic Dismembrator Model 500; 5 spulse on and 10 s off, total 21 min at 50% amplitude). Aftercentrifugation, the target proteins in the supernatants were purified.

Three approaches were used to purify the various recombinant proteins.His-tagged proteins were purified by the Profinity IMAC Ni-Charged Resin(Bio-Rad, Hercules, Calif., USA). Fusion proteins containing acellulose-binding module (CBM) and self-cleavage intein were purifiedthrough high-affinity adsorption on a large surface-area regeneratedamorphous cellulose. Heat precipitation at 70-95° C. for 5-30 min wasused to purify hyperthermostable enzymes. The purity of the recombinantproteins was examined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). A6PE was purified with 80 μM CoCl₂ presentin growth media, elution buffers, dialysis buffer, and protein storagebuffer.

Enzymes Used and their Activity Assays

Alpha-glucan phosphorylase (αGP) from Thermotoga maritima (UNIPROT IDG4FEH8) was used. Activity was assayed in 50 mM sodium phosphate buffer(pH 7.2) containing 1 mM MgCl₂, 5 mM DTT, and 30 mM maltodextrin at 50°C. The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO) (Vivaproducts, Inc., Littleton, Mass., USA).Glucose 1-phosphate (G1P) was measured using a glucose hexokinase/G6PDHassay kit (Sigma Aldrich, Catalog No. GAHK20-1KT) supplemented with 25U/mL phosphoglucomutase. A unit (U) is described as μmol/min.

Phosphoglucomutase (PGM) from Thermococcus kodakaraensis (UNIPROT IDQ68BJ6) was used. Activity was measured in 50 mM HEPES buffer (pH 7.2)containing 5 mM MgCl₂ and 5 mM G1P at 50° C. The reaction was stoppedvia filtration of enzyme with a Vivaspin 2 concentrator (10,000 MWCO).The product glucose 6-phosphate (G6P) was determined using ahexokinase/G6PDH assay kit (Sigma Aldrich, Catalog No. GAHK20-1KT).

Two different sources of phosphoglucoisomerase (PGI) were used fromClostridium thermocellum (UNIPROT ID A3DBX9) and Thermus thermophilus(UNIPROT ID Q5SLL6). Activity was measured in 50 mM HEPES buffer (pH7.2) containing 5 mM MgCl₂ and 10 mM G6P at 50° C. The reaction wasstopped via filtration of enzyme with a Vivaspin 2 concentrator (10,000MWCO). The product, fructose 6-phosphate (F6P), was determined using afructose 6-phosphate kinase (F6PK)/pyruvate dehydrogenase (PK)/lactatedehydrogenase (LD) coupled enzyme assay where a decrease in absorbanceat 340 nm indicates production of F6P. This 200 μL reaction contained 50mM HEPES (pH 7.2), 5 mM MgCl₂, 10 mM G6P, 1.5 mM ATP, 1.5 mM phosphoenolpyruvate, 200 μM NADH, 0.1 U PGI, 5 U PK, and 5 U LD.

Allulose 6-phosphate 3-epimerase (A6PE) from Thermoanaerobacteriumthermosaccharolyticum (UNIPROT ID D9TQJ4), SEQ ID NO 3, was used.Activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mMMgCl₂, 80 μM CoCl₂, 1 U/mL A6PP, and 10 mM F6P at 50° C. The reactionwas stopped via filtration of enzyme with a Vivaspin 2 concentrator(10,000 MWCO). The product, allulose 6-phosphate (A6P), was determinedusing allulose 6-phosphate phosphatase and detecting free phosphaterelease. To detect free phosphate release, 500 μL of a solutioncontaining 0.1 M zinc acetate and 2 mM ammonium molybdate (pH 5) wasadded to 50 μL of reaction. This was mixed and followed by 125 μL of 5%ascorbic acid (pH 5). This solution was mixed then incubated at 30° C.for 20 min. The absorbance at 850 nm was read to determine freephosphate release.

Allulose 6-phosphate phosphatase (A6PP) from Clostridium thermocellum(UNIPROT ID A3DC21), SEQ ID NO 9, was used. Activity was measured in 50mM HEPES buffer (pH 7.2) containing 5 mM MgCl₂, 80 μM CoCl₂, 1 U/mLA6PE, and 10 mM F6P at 50° C. The reaction was stopped via filtration ofenzyme with a Vivaspin 2 concentrator (10,000 MWCO). The product,allulose, was determined through detecting free phosphate release asdescribed for A6PE.

The recombinant cellodextrin phosphorylase and cellobiose phosphorylasefrom C. thermocellum are described in Ye et al. Spontaneous high-yieldproduction of hydrogen from cellulosic materials and water catalyzed byenzyme cocktails. ChemSusChem 2009; 2:149-152. Their activities wereassayed as described.

The recombinant polyphosphate glucokinase from Thermobifida fusca YX isdescribed in Liao et al., One-step purification and immobilization ofthermophilic polyphosphate glucokinase from Thermobifida fusca YX:glucose-6-phosphate generation without ATP. Appl. Microbiol. Biotechnol.2012; 93:1109-1117. Its activities were assayed as described.

The recombinant isoamylase from Sulfolobus tokodaii is described inCheng et al., Doubling power output of starch biobattery treated by themost thermostable isoamylase from an archaeon Sulfolobus tokodaii.Scientific Reports 2015; 5:13184. Its activities were assayed asdescribed.

The recombinant 4-alpha-glucanoltransferase from Thermococcus litoralisis described in Jeon et al. 4-α-Glucanotransferase from theHyperthermophilic Archaeon Thermococcus Litoralis. Eur. J. Biochem.1997; 248:171-178. Its activity was measured as described.

Sucrose phosphorylase from Caldithrix abyssi (UNIPROT H1XT50) was used.Its activity was measured in 50 mM HEPES buffer (pH 7.5) containing 10mM sucrose and 12 mM organic phosphate. Glucose 1-phosphate (G1P) wasmeasured using a glucose hexokinase/G6PDH assay kit supplemented with 25U/mL phosphoglucomutase as with alpha-glucan phosphorylase.

Enzyme units used in each Example below can be increased or decreased toadjust the reaction time as desired. For example, if one wanted toperform Example 9 in 8 h instead of 24 h, the units of the enzymes wouldbe increased about 3-fold. Conversely, if one wanted perform example 9in 48 h instead of 24 h the enzyme units could be decreased about2-fold. These examples illustrate how the amount of enzyme units can beused to increase or decrease reaction time while maintaining constantproductivity.

Example 1

To validate the technical feasibility of the enzymatic biosynthesis offructose 6-phosphate from starch, three enzymes were recombinantlyexpressed: alpha-glucan phosphorylase from T. maritima (UNIPROT IDG4FEH8), phosphoglucomutase from Thermococcus kodakaraensis (UNIPROT IDQ68BJ6), and phosphoglucoisomerase from Clostridium thermocellum(UNIPROT ID A3DBX9). The recombinant proteins were over-expressed in E.coli BL21 (DE3) and purified as described above.

A 0.20 mL reaction mixture containing 10 g/L soluble starch, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 0.5 mM ZnCl₂, 0.01 U ofαGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours.The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), wasdetermined using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P asdescribed above. The final concentration of F6P after 24 hours was 3.6g/L.

Example 2

Same tests as in Example 1 (other than reaction temperatures) werecarried out from 40 to 80° C. It was found that 10 g/L soluble starchproduced 0.9 g/L F6P at 40° C. and 3.6 g/L F6P at 80° C. after 40 hourreactions. These results suggest that increasing reaction temperaturefor this set of enzymes increased F6P yields, but too high temperaturemay impair some enzyme activity.

Example 3

It was found that, at 80° C., an enzyme unit ratio of αGP:PGM:PGI ofapproximately 1:1:1 resulted in fast F6P generation. It was noted thatthe enzyme ratio did not influence final F6P concentration greatly ifthe reaction time was long enough. However, the enzyme ratio affectsreaction rates and the total cost of enzymes used in the system.

Example 4

A 0.20 mL reaction mixture containing 10 g/L maltodextrin, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 0.5 mM ZnCl₂, 0.01 U ofαGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours.The reaction was stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), wasdetermined using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P asdescribed above. The final concentration of F6P after 24 hours was 3.6g/L.

Example 5

To test for F6P production from Avicel, Sigma cellulase was used tohydrolyze cellulose at 50° C. To remove beta-glucosidase from commercialcellulase, 10 filter paper units/mL of cellulase was mixed to 10 g/LAvicel at an ice-water bath for 10 min. After centrifugation at 4° C.,the supernatant containing beta-glucosidase was decanted. Avicel thatwas bound with cellulase containing endoglucanase and cellobiohydrolasewas resuspended in a citrate buffer (pH 4.8) for hydrolysis at 50° C.for three days. The cellulose hydrolysate was mixed with 5 U/mLcellodextrin phosphorylase, 5 U/L cellobiose phosphorylase, 5 U/mL ofαGP, 5 U/mL PGM, and 5 U/mL PGI in a 100 mM HEPES buffer (pH 7.2)containing 10 mM phosphate, 5 mM MgCl₂ and 0.5 mM ZnCl₂. The reactionwas conducted at 60° C. for 72 hours and high concentrations of F6P werefound (small amounts of glucose and no cellobiose). F6P was detectedusing the coupled enzyme assay described above. Glucose was detectedusing a hexokinase/G6PDH assay kit as described above.

Example 6

To increase F6P yields from Avicel, Avicel was pretreated withconcentrated phosphoric acid to produce amorphous cellulose (RAC), asdescribed in Zhang et al. A transition from cellulose swelling tocellulose dissolution by o-phosphoric acid: evidence from enzymatichydrolysis and supramolecular structure. Biomacromolecules 2006;7:644-648. To remove beta-glucosidase from commercial cellulase, 10filter paper units/mL of cellulase was mixed with 10 g/L RAC in anice-water bath for 5 min. After centrifugation at 4° C., the supernatantcontaining beta-glucosidase was decanted. The RAC that was bound withcellulase containing endoglucanase and cellobiohydrolase was resuspendedin a citrate buffer (pH 4.8) for hydrolysis at 50° C. for 12 hours. TheRAC hydrolysate was mixed with 5 U/mL cellodextrin phosphorylase, 5 U/Lcellobiose phosphorylase, 5 U/mL of αGP, 5 U/mL PGM, and 5 U/mL PGI in a100 mM HEPES buffer (pH 7.2) containing 10 mM phosphate, 5 mM MgCl₂ and0.5 mM ZnCl₂. The reaction was conducted at 60° C. for 72 hours. Highconcentrations of F6P and glucose were recovered because no enzymes wereadded to convert glucose to F6P. F6P was detected using the coupledenzyme assay described above. Glucose was detected using ahexokinase/G6PDH assay kit as described above.

Example 7

To further increase F6P yields from RAC, polyphosphate glucokinase andpolyphosphate were added. To remove beta-glucosidase from commercialcellulase, 10 filter paper units/mL of cellulase was mixed with 10 g/LRAC in an ice-water bath for 5 min. After centrifugation at 4° C., thesupernatant containing beta-glucosidase was decanted. The RAC that wasbound with cellulase containing endoglucanase and cellobiohydrolase wasre-suspended in a citrate buffer (pH 4.8) for hydrolysis at 50° C. wasincubated in a citrate buffer (pH 4.8) for hydrolysis at 50° C. for 12hours. The RAC hydrolysate was mixed with 5 U/mL polyphosphateglucokinase, 5 U/mL cellodextrin phosphorylase, 5 U/mL cellobiosephosphorylase, 5 U/mL of αGP, 5 U/mL PGM, and 5 U/mL PGI in a 100 mMHEPES buffer (pH 7.2) containing 50 mM polyphosphate, 10 mM phosphate, 5mM MgCl₂ and 0.5 mM ZnCl₂. The reaction was conducted at 50° C. for 72hours. F6P was found in high concentrations with only small amounts ofglucose now present. F6P was detected using the coupled enzyme assaydescribed above. Glucose was detected using a hexokinase/G6PDH assay kitas described above.

Example 8

To validate allulose production from F6P, 2 g/L F6P was mixed with 1U/ml A6PE and 1 U/ml A6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mMMgCl₂ and 80 μM CoCl₂. The reaction was incubated for 6 hours at 50° C.99% conversion of F6P to allulose was seen via HPLC (Agilent 1100series) using an Agilent Hi-Plex H-column and refractive index detector.The sample was run in 5 mM H₂SO₄ at 0.6 mL/min.

Example 9

To validate production of allulose from maltodextrin, a 0.20 mL reactionmixture containing 20 g/L maltodextrin, 50 mM phosphate buffered salinepH 7.2, 5 mM MgCl₂, 80 μM CoCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI,0.05 U A6PE and 0.05 U A6PP is incubated at 50° C. for 24 hours. Thereaction is stopped via filtration of enzyme with a Vivaspin 2concentrator (10,000 MWCO). Allulose is detected and quantified using anAgilent 1100 series HPLC with refractive index detector and an AgilentHi-Plex H-column. The mobile phase is 5 mM H₂SO₄, which runs at 0.6mL/min. Standards of various concentrations of allulose are used toquantify our yield.

Example 10

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 5.5), 5 mM MgCl₂, 80 μM CoCl₂, and 0.1 g/L isoamylase is incubatedat 80° C. for 24 hours. This is used to create another reaction mixturecontaining 20 g/L isoamylase treated maltodextrin, 50 mM phosphatebuffered saline pH 7.2, 5 mM MgCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 UPGI, 0.05 U A6PE, and 0.05 U A6PP is incubated at 50° C. for 24 hours.Production of allulose is quantified as in Example 9.

Example 11

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer(pH 4.5), 5 mM MgCl₂, and 1:200 dilution of Novozymes D6 pullulanase isincubated at 50° C. for 4 hours. This is used to create another reactionmixture containing 20 g/L pullulanase treated maltodextrin, 50 mMphosphate buffered saline pH 7.2, 5 mM MgCl₂, 80 μM CoCl₂, 0.05 U ofαGP, 0.05 U PGM, 0.05 U PGI, 0.05 U A6PE, and 0.05 U A6PP is incubatedat 50° C. for 24 hours. Production of allulose is quantified as inExample 9.

Example 12

To further increase allulose yields from maltodextrin, 0.05 U 4-glucantransferase (4GT) is added to the reaction described in example 9.

A 0.2 mL reaction mixture containing 20 g/L isoamylase treatedmaltodextrin (see example 9), 50 mM phosphate buffered saline pH 7.2, 5mM MgCl₂, 80 μM CoCl₂, 0.05 U of αGP, 0.05 U PGM, 0.05 U PGI, 0.05 UA6PE, 0.05 U A6PP, and 0.05 U 4GT is incubated at 50° C. for 24 hours.Production of allulose is quantified as in Example 9.

Example 13

To determine the concentration range of phosphate buffered saline (PBS),a 0.20 mL reaction mixture containing 50 g/L maltodextrin; 6.25 mM, 12.5mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline pH 7.2; 5 mMMgCl₂; 0.1 U of aGP; 0.1 U PGM; and 0.1 U PGI is incubated at 50° C. for6 hours. The short duration ensures completion is not reached, andtherefore differences in efficiency can be clearly seen. Production ofF6P is quantified using a fructose 6-phosphate kinase (F6PK)/pyruvatedehydrogenase (PK)/lactate dehydrogenase (LD) coupled enzyme assay wherea decrease in absorbance at 340 nm indicates production of F6P.Respectively, a yield of 4.5 g/L, 5.1 g/L, 5.6 g/L, 4.8 g/L, or 4.9 g/LF6P is obtained for the reactions containing either 6.25 mM, 12.5 mM, 25mM, 37.5 mM, or 50 mM phosphate buffered saline pH 7.2 (Table 1). Theseresults indicate that a concentration of 25 mM PBS pH 7.2 is ideal forthese particular reaction conditions. It is important to note that eventhe use of 6.25 mM PBS at pH 7.2 results in significant turnover due tophosphate recycling. This shows that the disclosed phosphate recyclingmethods are able to keep phosphate levels low even at industrial levelsof volumetric productivity (e.g., 200-300 g/L maltodextrin).

TABLE 1 Concentration of PBS pH 7.2 (mM) g/L of F6P 6.25 4.5 12.5 5.1 255.6 37.5 4.8 50 4.9

Example 14

To determine the pH range of the cascade reaction, a 0.20 mL reactionmixture containing 50 g/L maltodextrin; 50 mM phosphate buffered salinepH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 7.2, or 7.3; 5 mM MgCl₂; 0.02 U of αGP;0.02 U PGM; and 0.02 U PGI is incubated at 50° C. for 16 hours. Theunits are lowered to ensure completion is not reached, and thereforedifferences in efficiency can be clearly seen. Production of F6P isquantified as in example 12. Respectively, a yield of 4.0 g/L, 4.1 g/L4.2 g/L, 4.1 g/L, 4.4 g/L, 4.1 g/L, 3.8 g/L or 4.0 g/L F6P was obtainedfor reactions containing 50 mM phosphate buffered saline at pH 6.0, 6.2,6.4, 6.6, 6.8, 7.0, 7.2, or 7.3 (Table 2). These results indicate that apH of 6.8 is ideal for these particular reaction conditions, althoughthe system works through a wide pH range.

TABLE 2 pH of PBS g/L of F6P 6.0 4.0 6.2 4.1 6.4 4.2 6.6 4.1 6.8 4.4 7.04.1 7.2 3.8 7.3 4.0

Example 15

To investigate scale-up, a 20 mL reaction mixture containing 50 g/Lisoamylase treated maltodextrin (see Example 10), 50 mM phosphatebuffered saline pH 7.2, 5 mM MgCl2, 80 μM CoCl₂, 10 U of αGP, 10 U PGM,10 U PGI, 10 U A6PE, and 10 U A6PP is incubated at 50° C. for 24 hours.Production of allulose was quantified as in Example 9.

Example 16

To further increase allulose yields from maltodextrin, 0.05 U maltosephosphorylase is added to the reaction described in Example 9.

Example 17

To further increase allulose yields from maltodextrin, 0.05 Upolyphosphate glucokinase and 75 mM polyphosphate is added to thereaction described in Example 9.

Example 18

To produce allulose from fructose, a reaction mixture containing 10 g/Lfructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 80μM CoCl₂, 0.05 U fructose polyphosphate kinase, 0.05 U A6PE, and 0.05 UA6PP is incubated at 50° C. for 24 hours. Production of allulose isquantified as in Example 9.

Example 19

To produce allulose from glucose, a reaction mixture containing 10 g/Lglucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl₂, 80CoCl₂, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U A6PE, and0.05 U A6PP is incubated at 50° C. for 24 hours. Production of alluloseis quantified as in Example 9.

Example 20

To produce allulose from sucrose, a reaction mixture containing 10 g/Lsucrose, 50 mM phosphate buffered saline pH 7.0, 5 mM MgCl₂, 80 μMCoCl₂, 0.05 U sucrose phosphorylase, 0.05 PGM, 0.05 U PGI, 0.05 U A6PE,and 0.05 U A6PP is incubated at 50° C. for 24 hours. Production ofallulose is quantified as in Example 9.

Example 21

To further increase yields of allulose from sucrose, 75 mM polyphosphateand 0.05 polyphosphate fructokinase is added to the reaction mixture inexample 20. Production of allulose is quantified as in Example 9.

What is claimed is:
 1. An enzymatic process for preparing D-allulosefrom a starch derivative, the process comprising the steps of: (i)converting the starch derivative to glucose 1-phosphate (G1P), catalyzedby an alpha glucan phosphorylase (αGP); (ii) converting the G1P toglucose 6-phosphate (G6P), catalyzed by a phosphoglucomutase (PGM);(iii) converting the G6P to fructose 6-phosphate (F6P), catalyzed by aphosphoglucoisomerase (PGI); (iv) converting the F6P to allulose6-phosphate (A6P), catalyzed by an allulose 6-phosphate epimerase(A6PE); and (v) converting the A6P to D-allulose, catalyzed by anallulose 6-phosphate phosphatase (A6PP), wherein the A6PPdephosphorylate the A6P to produce the D-allulose and phosphate ions. 2.The process of claim 1, wherein the starch derivative is prepared by:enzymatic hydrolysis of starch, catalyzed by an isoamylase, apullulanase, an alpha-amylase, or a combination thereof; or acidhydrolysis of starch.
 3. The process of claim 1, wherein the processsteps are conducted under at least one of the following processconditions: at a temperature ranging from 37° C. to 85° C.; and/or at apH ranging from 5.0 to 8.0.
 4. The process of claim 1, wherein theprocess steps are conducted under at least one of the following processconditions: without adenosine triphosphate (ATP) as a source ofphosphate; without nicotinamide adenosine dinucleotide (NAD); and at aphosphate concentration from about 0.1 mM to about 150 mM, wherein:phosphate is recycled; wherein the step (v) involves an energeticallyfavorable chemical reaction; and wherein the steps (i) to (v) areconducted in a single reaction vessel.
 5. The process of claim 1,wherein the phosphate ions produced by A6PP dephosphorylation of the A6Pare used in the process step of converting the starch derivative to G1P.6. The process of claim 1, wherein the D-allulose is separated fromreaction components and degradation products of the steps (i-v) withoutusing chromatography.
 7. The process of claim 1, wherein the starchderivative is selected from the group consisting of amylose,amylopectin, soluble starch, amylodextrin, maltotriose, maltodextrin,maltose, and glucose.